Systemic Granulomatosis in the Meagre Argyrosomus regius: Fishing for a Plausible Etiology

Abstract

Meagre (Argyrosomus regius) is one of the fast-growing species considered for sustainable aquaculture development along the Mediterranean and Eastern Atlantic coasts. The emergence of Systemic Granulomatosis (SG), a disease marked by multiple granulomas in various tissues, poses a significant challenge in meagre aquaculture. In the current study, we investigate the association of Mycobacterium spp. and SG in offshore aquaculture facilities in Sardinia, Italy. A total of 34 adult seemingly healthy meagre were arbitrarily collected and analyzed, combining histological, microbiological, molecular, metagenomics, and in situ techniques to investigate the presence of pathogens. Ziehl-Neelsen (ZN), periodic acid-schiff (PAS), and Giemsa stains were performed for the detection of acid-fast bacteria, common parasites, and fungi within granulomas, respectively. Granulomas were detected in 91% (31/34) of fish. The affected organs were kidney (88%), liver (47%), heart (41%), intestine (17.6%), and brain (5%). Acid-fast staining, along with Mycobacterium spp. specific quantitative PCR (qPCR), in situ hybridization (ISH) assay, and microbiological analyses showed negative results for the detection of Mycobacterium spp. and other bacteria implicated in granuloma formation. However, PCR amplification and sequencing of the 65-kDa heat shock protein gene revealed the presence of M. chelonae in 13% of both formalin-fixed and frozen liver tissues. Bacterial isolation failed to detect nontuberculous mycobacteria (NTM) and other bacteria typically associated with granulomas. Consistently, the use of an M. chelonae-specific probe in ISH failed to identify this bacterial species in granulomas. Collectively, results do not support the role of M. chelonae in the development of granulomas and suggest rejecting the hypothesis of a potential link between NTM and SG.

Keywords: Mycobacterium chelonae; fish aquaculture; granulomas; in situ hybridization assay.

Figure 1

(a) Affected meagre showing severe hemorrhages and bilateral exophthalmia. (b) Heart of meagre with visible white nodules (red arrows) on the epicardium. Bar: 0.2 cm.

Figure 2

(a) Multifocal granulomas in the heart (Hematoxylin and eosin—H&E. Bar: 100 µm). (b) Multifocal granulomas in the liver (H&E. Bar: 100 µm). (c) High power field of a granuloma in the kidney characterized by a necrotic hypereosinophilic center (asterisk) surrounded by epithelioid and spindle cells arranged in concentric layers (arrows) (H&E. Bar: 20 µm). (d) Negative Ziehl–Neelsen stain of a granuloma in the kidney (ZN. Bar: 20 µm). (e) Focal granuloma in the brain (thick arrows) (H&E. Bar: 100 µm).

Figure 3

Bar chart illustrating the relative abundance (in percentage) of the main microbial taxa at the phylum level in the brain, heart, spleen, kidney, and intestine of a meagre affected by systemic granulomatosis.

Figure 4

Maximum Likelihood tree shows the hsp65 sequences obtained from systemic granulomatosis-affected meagre cluster with the Mycobacterium chelonae. Nocardia farcinica was selected as the outgroup. The other sequences are the most probable species causing mycobacteriosis in fish. Evolutionary analyses were conducted in MEGA7, performed using Maximum Likelihood method, Tamura–Nei model, and bootstrap 1000 replicates.

Figure 5

(a) Granuloma in meagre’s liver without in situ hybridization signals (Hematoxylin counterstain. Bar: 20 µm). (b) Numerous, 1–2 microns in length, bacillary red rods (red chromogen) inside a granuloma of a Carassius auratus experimentally infected with M. chelonae (Hematoxylin counterstain. Bar: 20 µm).